Accurate quantitation of RNA and DNA by competetitive polymerase chain reaction

Abstract

A process for quantitating nucleic acids species in a sample is described which comprises co-amplification of a competitive template with the sample template wherein the competitive template utilizes primers identical to those utilized by the sample template and wherein the competitive template is distinguishable from the sample template.

What is claimed is: 
     
       1. A method for determining the amount of a target nucleic acid sequence in a sample, said method comprising (a) co-amplification of a target nucleic acid sequence and a known amount of a standard nucleic acid sequence in the same assay sample, wherein said target nucleic acid sequence and said standard nucleic acid sequence bind primers consisting essentially of the same sequence during said co-amplification and wherein said standard nucleic acid sequence differs from said target nucleic acid sequence such that either a restriction enzyme recognition site is created or destroyed, or sufficient contiguous bases are added to or deleted from said target nucleic acid sequence such that the size difference between said target nucleic acid sequence and said standard nucleic acid sequence can be detected by gel electrophoresis;   (b) detecting products of said co-amplification using gel electrophoresis; and   (c) determining the amount of said target nucleic acid sequence in said sample.   
     
     
       2. A method for determining the amount of a target nucleic acid sequence in a sample, said method comprising: (a) adding a known amount of a standard nucleic acid sequence to a sample that contains said target nucleic acid sequence, wherein said standard nucleic acid sequence and said target nucleic acid sequence bind amplification primers consisting essentially of the same sequence, and wherein said standard nucleic acid sequence differs from said target nucleic acid sequence such that either a restriction enzyme recognition site is either created or destroyed or sufficient contiguous bases are added to or deleted from said target nucleic acid sequence such that the size difference between said target nucleic acid sequence and said standard nucleic acid sequence can be detected by gel electrophoresis;   (b) adding said amplification primers to said sample;   (c) co-amplifying said target nucleic acid sequence and said standard nucleic acid sequence under conditions wherein said standard nucleic acid sequence and said target acid sequence bind said amplification primers;   (d) detecting products of said co-amplifications using gel electrophoresis; and   (e) determining the amount of said target nucleic acid sequence in said sample from the amount of amplification of said standard nucleic acid sequence.   
     
     
       3. The method of any one of claims 1 or 2, wherein said nucleic acid sequence is DNA. 
     
     
       4. The method of any one of claims 1 or 2, wherein said nucleic acid sequence is RNA. 
     
     
       5. The method of claim 4, wherein said RNA is mRNA. 
     
     
       6. The method of any one of claims 1 or 2, wherein said amplification primers consist of the same sequence. 
     
     
       7. The method of any one of claims 1 or 2, wherein said standard nucleic acid sequence comprises a genomic sequence of said target template. 
     
     
       8. The method of any one of claims 1 or 2, wherein said standard nucleic acid sequence is a mutation of said target template. 
     
     
       9. The method of claim 8, wherein said mutation is the addition of a restriction enzyme site. 
     
     
       10. The method of claim 8, wherein said mutation is the removal of a restriction enzyme site. 
     
     
       11. The method of claim 8, wherein said mutation is the addition of an intron sequence. 
     
     
       12. The method of claim 11, wherein said intron is 100-200 bp. 
     
     
       13. The method of claim 8, wherein said mutation is the removal of an intron sequence. 
     
     
       14. The method of any one of claim 1 or 2, wherein said primer is a 20-40 mer. 
     
     
       15. The method of any one of claims 1 or 2, wherein said primer contains 50-60% GC content. 
     
     
       16. The method of any one of claims 1 or 2, wherein the amplification of said nucleic acid sequences result in an amplified product of 200-266 bp.